A gene amplification method, which is an in vitro diagnostic testing (IVD testing) technology of amplifying gene having a specific sequence to determine the presence and absence of gene, has been used in various fields such as a food test, a genetically modified organism (GMO) test as well as a pathogenic microorganism test in various animals, plants, or the like, in addition to humans, and a genotype test. In order to perform an accurate gene amplification test from various biological samples, first, a nucleic acid extraction process of removing various reaction inhibitors included in biological samples and inhibiting a gene amplification reaction from the biological samples and obtaining a high purity target nucleic acid is required. Here, the target nucleic acid may be DNA, RNA, or a mixture thereof according to the detection object. The gene amplification test is completed by mixing the target nucleic acid extracted as described above with a gene amplification solution to perform the gene amplification reaction and confirming a DNA corresponding to a DNA length of a genetically amplified product through electrophoresis.
As used herein, the term “biological samples”, which are materials from living things, may be interpreted as a meaning including all of materials defined as living things as well as animals, plants, microorganisms, virus, and fungi.
As a method for amplifying DNA in the gene amplification test, a PCR method has been mainly used. In order to detect a trace amount of DNA, a nested PCR method for performing a PCR reaction once more using a complementary primer in a primer base sequence of the amplified DNA has been mainly used. In order to test an expression amount of mRNA of RNA virus or a specific gene, a reverse transcription-PCR (RT-PCR) has been used. In addition, various methods for amplifying DNA or RNA at a predetermined temperature without performing a thermal cycling reaction have been developed.
Meanwhile, since in this PCR method, when DNA amplification is performed in some degree, deoxynucleotide triphosphate is exhausted, such that DNA amplification reaches a limitation point at which amplification may not be performed any more, there is a limitation in quantitative analysis. For example, in the case in which a template nucleic acid has a high initial concentration, the template nucleic acid becomes saturated in a short reaction of 20 cycles or less, and in the case in which a template nucleic acid has an initial concentration of 1/1000 times the concentration in the above-mentioned case, the template nucleic acid becomes saturated after at least 30 cycles, but after 30 cycles, in both of the cases, detection amounts are the same as each other. In order to solve this problem, a quantitative real-time PCR technology capable of accurately and quantitatively measuring an initial concentration of a nucleic acid by measuring a concentration of the nucleic acid after each of the cycles to measure a cycle at which a concentration of the nucleic acid reaches a predetermined concentration. Since this technology may quantitatively measure a concentration of virus or pathogenic bacteria, this method has been developed as a significantly useful technology in view of molecular diagnosis. In the quantitative real-time PCR technology, methods using fluorescence increased in proportion to an amount of DNA have been mainly used. The fluorescence method as described above is divided into an amplified DNA sequence specific method and an amplified DNA sequence non-specific method. The amplified DNA sequence non-specific method is a method using an intercalating dye binding all of the amplified DNA(s) to increase fluorescence. In case of using this method, an amount of fluorescence is increased in proportion to the amount of all of the amplified DNA(s). Therefore, in the case in which a non-specific amplified product such as a primer-dimer is formed or in the case of amplifying which at least two specific targets, it is impossible to accurately detect an initial amount of the target nucleic acid. On the other hand, in methods using a fluorescent probe specific to an amplified DNA sequence, a plurality of target nucleic acids are amplified in one tube and at the same time, multiplex quantitative detection of nucleic acids may be performed. In this method, since various kinds of probes having different fluorescent properties are selectively hybridized in each of the amplified DNA(s) to thereby exhibit fluorescence while amplifying targets using a pair of primer, a multiplex quantitative real-time PCR method of detecting each of the fluorescent products to quantitatively measuring each of the products has been developed. However, in this method, since as the number of target nucleic acids is increased, a pair of primers and a probe, that is, triple kinds of oligonucleotides should be injected per each target nucleic acid, there was a problem in that in the case of performing the multiplex quantitative real-time PCR on four or more targets, performance may be rapidly decreased. On the other hand, since in a general multiplex PCR, multiplex PCR of ten kinds of target nucleic acids may be optimized so as to be suitably performed, the multiplex PCR may be advantageous for detecting several kinds of target nucleic acid. However, since this PCR method is not quantitative, a method for quantitatively testing a multiplex target has been required. As another limitation of the quantitative real-time PCR method, there was a problem in that since it is impossible to confirm a length of the amplified DNA, it was impossible to use the quantitative real-time PCR method in order to analyze presence or absence of DNA deletion or DNA insertion or detect the number of repeated base such as variable numbers of tandem repeat (VNTR).
In order to solve this problem, an object of the present invention is to provide an automatic apparatus capable of performing quantitative real-time PCR and then confirming a length of the amplified product using a general PCR. Therefore, an object of the present invention is to provide an apparatus capable of simultaneously amplifying various targets, simultaneously and quantitatively each of the targets, and quantitatively measuring an initial amount of DNA in the corresponding target simultaneously with analyzing presence or absence of DNA deletion or DNA insertion or detecting the number of the repeated base such as VNTR.
Meanwhile, since in the gene amplification test, detection may be performed with high sensitivity and specificity, the gene amplification test has been variously used in testing various microorganisms and genes. However, there is an amplicon contamination problem that even though a trace amount of aerosol including the genetically amplified product (amplicon) is contaminated, a false-positive result is obtained due to high sensitivity. In order solve this problem, methods for inactivating the amplified product using ultraviolet rays or an enzyme reaction have been developed. The method using ultraviolet rays is a method for converting a contaminated product into non-amplified DNA by performing an amplification reaction after mixing 8-methoxypsoralene (8-MOP) with a PCR reaction solution in advance, irradiating ultraviolet rays to perform a photochemical reaction with DNA, even in the case in which a PCR product is contaminated later. In the method using an enzyme, which is a method using deoxyuridine triphosphates (dUTP) and uracil deoxy-glycosidase (UDG) enzyme, dUTP is added to a PCR reaction solution, such that a PCR product includes a deoxyuracil base. Then, in this method, the UDG enzyme is added to the PCR product, such that a contaminated PCR product is dissolved and removed through a UDG enzyme reaction before a PCR amplification reaction. However, in the case of the nest PCR in which PCR should be continuously performed using the PCR product, it is impossible to use these methods. Therefore, generally, in an organization performing the gene amplification test, separate laboratories for nucleic acid extraction and purification, preparation of gene amplification reaction sample, and electrophoresis analysis are prepared, respectively, and each of the works is preformed in these laboratories, such that it is costly to prepare and operate the gene amplification laboratories. Nevertheless, since there is still a risk of a false-positive result generated due to contamination by a worker and a false-negative result generated due to an error in nucleic acid extraction and gene amplification reaction solution preparation, a test operation method for preventing the false-positive result and false-negative result is complicated, such that the test is mainly performed in a large hospital and a special clinical test organization.
Therefore, in the gene amplification test, a demand for an economic apparatus for automatically amplifying genes capable of removing the above-mentioned possibility of false-positives and false-negatives and increasing accuracy, reproducibility, and efficiency of the test has been gradually increased.
In order to satisfy the above-mentioned demand, before describing the present invention, each of the steps for performing the gene amplification test according to the related art will be described.
As a method for extracting a nucleic acid from biological samples,
in general, a method using magnetic particles has been widely used. This method is a method for rapidly attaching target nucleic acids to fine magnetic particles having a wide surface area in a liquid suspension state, applying a magnetic field to coagulate the magnetic particles including the target nucleic acid attached thereto, removing the filtrate, washing the magnetic particles, eluting pure nucleic acid, and then purifying the nucleic acid, and automation equipments relating to this method have been variously developed.
Recently, an automated method using a pipette has been generally and widely used.
Various methods for separating magnetic particles using a disposable pipette form have been disclosed by Lab system Ltd. as in U.S. Pat. No. 5,647,994. This method is a related art relating to a method for coagulating magnetic particles in a pipette as in U.S. Pat. No. 5,702,950 or 6,187,270. The apparatus using the disposable pipette is composed of a component having a single tube shape, connected in series to a zet channel that is defined as a distal flow gate of the tube, and having a diameter smaller than that of a separation chamber; a magnetic component positioned at a first position adjacent to an outside of a separation wall or a second position in the separation chamber and arranged so that when the magnetic component is positioned at the first position, magnetic particles may be collected by an influence of a magnetic field, and when the magnetic component is positioned at the second position, it is impossible to catch the magnetic particles any more; and a tube, which is second portion connected in series to the separation chamber having a cylindrical shape and separated from the zet channel, having a structure in which a cylinder channel is equipped with a movable piston to thereby suck and discharge a liquid.
In addition, various purification apparatuses using a pipette and a magnetic property have been suggested.
However, in the cases of all of the structures as described above, a method capable of separating a target nucleic acid from a solution using a disposable pipette to suspend the separated target nucleic acid in a different solution has been suggested, but these structure have a large limitation in that nucleic acid extraction may be frequently unsuccessful due to a blocking phenomenon generated at a lower end portion of the pipette by magnetic particles.
Further, there are problems in that since a serial process of separating the target nucleic acid from a biochemical mixture solution is performed in the pipette, uniform suspension of the solution is difficult, and after the washing corresponding to a final step of purification is completed, since a washing solution is not completely removed but a residual washing solution is included at the time of elution, a subsequent gene amplification process, or the like, may be affected by the residual washing solution.
In the performing of the gene amplification reaction from the extracted target nucleic acid,
various methods such as PCR, nested PCR, RT-PCR, isothermal nucleic acid amplification method, and the like, have been developed in order to amplify the target nucleic acid. Generally, a preparation step of mixing an extracted target nucleic acid with a gene amplification reaction solution to prepare a gene amplification reactant and a reaction step of performing a reaction are required. In the preparation step of mixing a gene amplification reaction solution containing a primer, nucleic acid polymerase, a nucleotide triphosphate dNTP or NTP, which is a polymerization monomer, and a buffer with the extracted target nucleic acid at a predetermined amount to prepare the reaction. In the gene amplification reaction step, in the case of isothermal amplification, there is a need only to maintain a predetermined temperature, but in the case of using PCR, a heating and cooling step for a thermal cycling reaction is required. Since a temperature should be increased in order to perform the gene amplification reaction, generally, sealing is performed in order to prevent evaporation. However, in the case of sealing a gene amplification reaction vessel, the sealed vessel should be opened and the genetically amplified product should be transferred to an electrophoresis gel 362 in order to perform an electrophoresis analysis on the genetically amplified product after gene amplification reaction, but there are problems in that a complicated apparatus for automatically performing the process as described above is required, and the genetically amplified product may be contaminated. Particularly, in the case of nested PCR, since it is impossible to use an inactivation method of the amplified product for preventing contamination of the amplified product, the nest PCR depends on a space separation for performing the test.
In a process of analyzing the genetically amplified product through the electrophoresis,
generally, an agarose electrophoresis method has been used. This method, which is a traditional method, has advantages such as a cheap cost, and simple analysis, but there are problems in that an analysis time is relatively long, and a lot of manual work of those skilled in the art should be performed. Recently, in order to solve this problem, methods using a capillary electrophoresis capable of rapidly and automatically performing electrophoresis have been developed, but an apparatus and supplies are expensive, such that these methods have been restrictively used. Since in all of the electrophoresis steps, analysis is always performed using the genetically amplified product, there is a problem in that fine aerosol generated therefrom may be mixed again with the gene amplification reaction solution to cause false-positives. Therefore, a method capable of preventing this problem is required.
Various apparatuses are developed for quantitative real-time PCR. However, most of the apparatuses are complicated and expensive. In addition, an apparatus capable of continuously and automatically performing the quantitative real-time PCR as in a general PCR reaction has not been developed, such that a simple and convenient apparatus and method capable of continuously performing this process have been required.
In performing the entire process of nucleic acid extraction, quantitative real-time PCR/PCR, and electrophoresis as described above, an apparatus and method for automatically analyzing biological samples capable of preventing contamination by external and internal contamination factors, having high reliability, increasing convenience, reproducibility, and efficiency, and having excellent test accuracy by automating the entire process required for analysis while continuously performing quantitative real-time PCR and general PCR have been required.